Project Summary G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) and function to turn off G protein signaling and turn on arrestin-mediated signaling. GRKs have a modular structure with a central catalytic domain inserted into a regulator of G protein signaling homology (RH) domain, which itself is bracketed by an N-terminal ?-helical domain and a C-terminal lipid-binding region. X-ray crystallography has revealed that the RH and catalytic domains have extensive contacts with each other and help to maintain the kinase in an inactive open conformation. To gain mechanistic insight into GRK regulation of GPCRs, we have studied GRK5 interaction with the ?2-adrenergic receptor (?2AR). GRK5 has been implicated in several diseases including heart failure and hypertension and a GRK5-Q41L polymorphism prevalent in African Americans has an enhanced ability to desensitize the ?2AR and protects against the development of congestive heart failure. We have identified conditions to generate a ?2AR-GRK5 complex to gain initial insight on the interface of these proteins. Our data support a model that involves a receptor- mediated disruption of an ionic lock between the GRK5 RH and catalytic domains that is essential for receptor phosphorylation. We propose to utilize GRK5 interaction with the ?2AR as a model to further dissect the mechanisms involved in GRK activation and gain insight into the normal regulation and function of this important enzyme family. Our central hypothesis is that GRK structure and function is mechanistically linked to interaction with GPCRs. In aim 1, we will test our initial structural model by mutagenesis and further characterize the dynamics of ?2AR-GRK5 interaction using radiolytic footprinting, double electron-electron resonance spectroscopy and molecular dynamics simulations. In aim 2, we will use X-ray crystallography and single particle cryo-electron microscopy to provide insight into the ?2AR-GRK5 interface as well as the structural changes that the ?2AR and GRK5 undergo upon binding. We will confirm the binding interface of these proteins and the mechanism of activation using molecular and biochemical approaches. In aim 3, we will focus on the regulation of GRK5 by calmodulin. These studies are supported by a crystal structure of a calmodulin-GRK5 complex that reveals that calmodulin binding disrupts the ionic lock between the RH and catalytic domains and promotes closure of the catalytic domain. We will verify the calmodulin-GRK5 interface by mutagenesis, further dissect how calmodulin activates GRK5 and define the role of calmodulin on GRK5 localization and function in cells. Overall, these studies have broad significance for understanding GRKs and GRK-GPCR interaction and should facilitate the development of strategies to specifically regulate GRK function in the treatment of disease.